This temperature is commonly used for culture of S. agalactiae from fish [26]. Isolates were checked for Gram reaction and morphology and tested in a group B-specific latex agglutination test (Slidex Strepto Plus B; bioMérieux, Marcy L’Étoile, France). Single colonies were transferred to Brain Heart Infusion (BHI) broth (Oxoid, Basingstoke, United Kingdom) and incubated with gentle shaking at 28°C for 12h (ß-haemolytic strains, fast growing) or 48h (non-haemolytic strains, slow growing). AZD1390 Species identity of S. agalactiae was confirmed by polymerase chain reaction (PCR), using forward primer STRA-AgI (5′-AAGGAAACCTGCCATTTG-′3) and reverse primer STRA-AgII (5′-TTAACCTAGTTTCTTTAAAACTAGAA-3′),

which target the 16S to 23S rRNA intergenic spacer region [27]. Broth cultures were also used for PFGE as described below. Comparative typing: PFGE Bacterial cells were pelleted by centrifugation of 1 ml of incubated BHI, re-suspended in

0.5 ml of TE buffer (10 mM Tris-HCl, 1mM EDTA), warmed to 56°C and mixed with 0.5 ml of 2% (weight/vol) low-melting point agarose (Incert agarose; Lonza, Slough, United Kingdom) in TE buffer. The mixture was then pipetted into reusable plug moulds (Catalogue number 170-3622; BioRad Laboratories, Hemel Hempstead, United Kingdom) producing 20 × 9 × 1.2 mm3 agarose blocks. Each solidified plug was placed into 2 ml of TE buffer containing 4 mg of lysozyme (Sigma Aldrich, Poole, United Kingdom) (2 mg ml-1) and incubated overnight at 37°C with gentle shaking. The buffer was then Lumacaftor mw replaced with 2 ml of ES buffer (0.5 M EDTA–1% BMN 673 solubility dmso (weight/vol) N-lauroyl sarcosine [pH 8.0 to 9.3]) supplemented with 4 mg of proteinase K (Promega,

Southampton, United Kingdom) (2 mg ml-1) and incubated at 56°C for a minimum of 48 hr. Plugs were washed 6 times for 1 hr in TE buffer at room temperature and with gentle shaking. A slice (4 × 4 × 1.2 mm3) from each plug was exposed to digestion with restriction endonuclease SmaI (20 U in 100 μl of fresh reaction buffer; New England Biolabs, Hitchin, United Kingdom) at 25°C overnight. PFGE was performed with a CHEF-mapper system (BioRad Laboratories) in 0.5 × TBE using a 1% (weight/vol) agarose gel (Pulsed Field Certified Agarose, BioRad Laboratories), a run time of 24 hr and switch time of 3-55 s (linear ramp) at 14°C. Patterns were observed by UV transillumination after SYBR Gold staining (Invitrogen, Paisley, United Kingdom). Computer-assisted data analysis and dendogram construction were performed with Phoretix 1D Pro software (TotalLab Ltd, Newcastle upon Tyne, United Kingdom). Similarities between PFGE patterns were also assessed visually using standard criteria [10]. Housekeeping genes: multilocus sequence typing MLST consisted of the amplification by PCR and sequencing of seven housekeeping genes, namely adhP, atr, glcK, glnA, pheS, sdhA, and tkt[13].